Journal: Molecular and Cellular Biology
Article Title: In Vitro Reconstitution of the End Replication Problem
Figure Lengend Snippet: Analysis of terminal restriction fragments from replicated linear DNAs. (A and B) The bound fraction of pSVO11-bead replication products was purified and treated with either λ exonuclease or exonuclease III. To know the exonuclease digestion rates, we treated a separately prepared 199-bp terminal fragment with these exonucleases and found that under the employed conditions, approximately 100 nt is digested from the ends, albeit relatively asymmetrically (data not shown). After the digestion, a half aliquot of the DNA was further treated with Dra I, which produces 199- and 497-bp fragments from the left and right arms of the DNA, respectively (A). Samples were run in a 6% denaturing acrylamide gel, dried, and autoradiographed. Heavily and lightly exposed autoradiographs of the same gel are shown. Control pSVO11 DNA was digested with Bsr FI, and the two ends were filled-in with dNTPs. The resultant blunt-ended linear pSVO11 was first treated with either λ exonuclease or exonuclease III, followed by Dra I digestion. The products were first dephosphorylated by alkaline phosphatase at their 5′ ends and then labeled by T4 polynucleotide kinase and [γ- 32 P]ATP. Dra I digests DNA at a TTT/AAA site, leaving blunt ends. Therefore, the two 199-nt and 497-nt fragment strands have the same nucleotide lengths (arrows). However, because of the effect of different base compositions on migration rates, two distinct 199-nt single-stranded DNA bands are visible in lane 1. The upper and lower bands (marked by open and filled circles, respectively) of the 199-nt doublet were completely digested by λ exonuclease and exonuclease III, respectively (lanes 2 to 5). The 199- and 197-nt bands were detected in pSV011-band replication products. These two bands were resistant to λ exonuclease (lanes 9 and 11). In contrast, the 199-nt band was completely digested, and the 497-nt band was significantly trimmed by exonuclease III (lanes 13 and 15; shorter-sized 497-nt bands are indicated by a bracket). These results indicate that the observed 497- and 199-nt bands were derived solely from a strand whose 3′ ends correspond to nascent radiolabeled DNA ends. Several extra bands were observed in lane 7. We do not know the precise origin of these signals. However, because they are both λ exonuclease and exonuclease III sensitive, it is likely they represent unligated lagging strand DNA molecules derived from internal template regions. It seemed that λ exonuclease had reached the Dra I site on the template (cold) strand of some molecules, because the signal intensity of the 199-nt band decreased after the λ exonuclease treatment.
Article Snippet: The purified DNA was either treated with or without λ exonuclease (GIBCO), exonuclease III (Takara), and exonuclease I (New England BioLabs).
Techniques: Purification, Acrylamide Gel Assay, Labeling, Migration, Derivative Assay